Microfluidic droplet encapsulation

ABSTRACT

Microfluidic devices and methods for the encapsulation of particles within liquid droplets are disclosed. The new methods and devices form 1-100 picoliter-size monodisperse droplets containing the particles, such as single cells, encapsulated in individual liquid droplets. The particles can be encapsulated in droplets of a fluid by passing a fluid containing the particles through a high aspect-ratio microchannel to order the particles in the fluid, followed by forming the fluid into droplets. The resulting fraction of the liquid droplets with a single particle (e.g., a cell) is higher than the corresponding fraction of single-particle liquid droplets predicted by Poisson statistics.

RELATED APPLICATIONS

This application claims the benefit of U.S. provisional patentapplication Ser. No. 61/055,653, filed on May 23, 2008. The subjectmatter of this U.S. provisional patent application is incorporatedherein by reference in its entirety.

STATEMENT OF FEDERALLY SPONSORED RESEARCH

This application includes work supported by the NSF (DMR-0602684 andDBI-0649865). The United States government has certain rights in thisapplication.

TECHNICAL FIELD

This disclosure relates to the encapsulation of analyte material, suchas cells, in liquid droplets.

BACKGROUND

Microfluidic devices and methods promise breakthrough applications inbiotechnology such as directed evolution, tissue printing, andbead-based PCR in emulsions, while facilitating many quantitativestudies of biology at a single-cell level. For example, in somemicrofluidic methods, individual cells can be made to reside withinseparate picoliter-volume liquid drop, chemically isolated from otherdroplets. This can permit cell-secreted molecules to rapidly achievedetectable concentrations in a confined fluid droplet surrounding theencapsulated cell. Similarly, uptake of trace chemicals specific toindividual cells can be probed due to their depletion within theconfined extracellular fluid. Moreover, highly monodisperse droplets ofwater in an inert and immiscible carrier fluid can be formed at rates ofseveral kHz using microfluidic techniques. These droplets can becombined in pairs, split in two, and selected based on the contents ofindividual droplets. However, variability in the number of cells orother particles per drop of fluid due to stochastic cell loading is amajor barrier to an effective use of these techniques.

Existing processes for loading individual cells into droplets aretypically random processes with the distribution of the number of cellsin each droplet being dictated by Poisson statistics. Accordingly, theprobability of a drop containing k cells is λ^(k) exp(−λ)/(k!), where kis the average number of cells per drop. The ratio of dropletscontaining one cell to those containing two is 2/λ. This means that tominimize the number of droplets that contain more than a single cellrequires very low average loading densities. As a result, most dropletsactually contain no cells whatsoever. This constraint significantlyreduces the number of usable droplets. For example, only 15.6% of alldroplets will contain one cell if no more than one in ten of theoccupied droplets can be allowed to hold two or more cells. There is aneed for microfluidic devices and methods for forming a higherproportion of liquid droplets containing a single cell.

SUMMARY

Microfluidic devices and methods disclosed herein provide encapsulationof particles within liquid droplets, including formation ofpicoliter-size monodisperse droplets containing the particles. Byordering the particles in a fluid stream within a microfluidic channelbefore droplet formation, droplets containing a single particle can beformed. The particles can be living cells or other material derived froma biological fluid sample, such as blood, or synthetic materials, suchas polymeric beads. For example, fluid droplets containing a single cellcan be repeatedly generated in an aqueous fluid (e.g., a salinesolution). The invention is based, in part, on the discovery thatpassing a particle (e.g., solid analyte particles, or cells) rapidlythrough a high aspect-ratio microchannel and into a droplet generatorresults in the formation of a desirable fraction of liquid droplets witha single particle per droplet. In general, the microchannel dimensionsand fluid flow rate can be selected using criteria described herein toprovide a fluid stream of ordered particles substantially evenly spacedalong the length of the microchannel before entering the dropletgenerator. In addition, particles, e.g., cells, tend to enter thedroplet generator with the frequency of droplet formation. In theresulting droplets, the fraction of single-particle liquid droplets ishigher than the corresponding fraction of single-particle liquiddroplets predicted by Poisson statistics.

In one aspect, the invention features methods of encapsulating particlesin liquid droplets. The methods include passing a particle (e.g., cells)through a channel in a fluid medium and forming the fluid medium into aplurality of droplets. In one example, the largest particle has amaximum cross-sectional dimension that is at least about 10% (e.g., 10%to about 40%, including 10-40, 20-30, 25, 30, or 35%) of the smallestcross-sectional dimension of the channel through which the fluid passes.The plurality of particles in the fluid medium through a channel canhave a minimum cross-sectional dimension D, wherein the largestparticles in the plurality has a maximum cross-sectional dimension thatis at least about 0.1 D (e.g., about 0.1 D to 0.4 D). The fluid mediumcan be formed into a plurality of picoliter droplets containing kparticles outside the channel, wherein the proportion of the dropletscontaining k particles is greater than λ^(k) exp(−λ)/(k!) (i.e., theproportion given by random, Poisson statistics), when λ is the averagenumber of particles per droplet. For example, the proportion of dropletscontaining one particle is greater than λ^(k) exp(−λ), including aproportion of droplets containing one particle of at least about 0.9λ.The proportion of droplets containing only one particle can be at leastabout 90% of the total number of droplets. The particle in the fluidmedium leaving the channel to form the plurality of droplets preferablyincludes particles having a substantially uniform spacing with respectto adjacent particles in the direction of the fluid medium flow.

In certain of these methods, forming the fluid medium into a pluralityof droplets can include contacting the fluid medium with a second medium(e.g., an oil) immiscible in the first medium (e.g., an aqueoussolution) to form the droplets in the second medium. For example, thefluid medium can be passed through a nozzle or other droplet generatingdevice to form the plurality of droplets.

In another aspect, the invention features systems for encapsulatingparticles in a fluid medium. The systems can include a microfluidicchannel having a minimum cross-sectional dimension D adapted to receivea fluid medium containing a plurality of particles having a maximumindividual cross-sectional dimension of at least about 0.1 D, and adroplet generator in fluid communication with the microfluidic channel.The microfluidic channel is configured to passively order the particleswithin the fluid medium while passing through the microfluidic channel.For example, the microfluidic channel can have a dimension D that isless than 1 mm, including 10-100 micrometers, and can be straight orcurved. The droplet generator can include a nozzle at an outlet of themicrofluidic channel. The droplet generator can also include a vesselcontaining a second fluid medium that is immiscible in the fluid mediumexiting from the microfluidic channel. The droplet generator can producedroplets of the fluid medium in the second fluid medium outside of themicrofluidic channel.

The methods and devices described herein are useful, in particular, forloading particles into droplets for applications that demand minimalnumbers of empty droplets in addition to a high ratio of single-celldroplets to multiples. For example, the methods and devices can be usedin the creation of tissue engineered constructs by “printing” cells ontoa substrate as a spray of picoliter-size aqueous droplets in air willreceive a significant boost in resolution from this ability to controlcell loading, allowing the narrowest possible lines to become the widthof a single-cell. Such ordered encapsulation becomes even more importantfor applications where streams of droplets, each with single-particlesof two varieties, are combined to create larger droplets carryingexactly one particle of each kind; the number of suitable droplets forthe above conditions would rise from 0.15% without ordering to about80%, about 500 times higher.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skillsin the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

As used herein, unless otherwise indicated, the term “particle” refersto a small discrete mass of solid or liquid matter, such as a cell or asolid particle that can be discretely transported in a fluid stream.

As used herein, unless otherwise indicated, the term “fluid” refers to agas or liquid, such a liquid biological sample (e.g., whole blood).

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing parameters useful in predicting when particleordering can occur within a fluid passing through a microfluidicchannel.

FIGS. 2A-2D are a series of schematic diagrams showing the ordering ofparticles flowing through a straight microfluidic channel.

FIG. 3 is a schematic diagram showing the separation, ordering, andfocusing of particles in a fluid passing through a symmetrically curvedserpentine microfluidic channel.

FIG. 4A shows a series of time-averaged images showing the focusing ofcultured cells in a serpentine microfluidic channel at differentReynolds numbers based on the mean channel velocity (R_(e)) values from0.05 to 5.0. FIG. 4B is a graph showing the intensity cross sections atvarious turns and at the outlet of the microfluidic channel shown inFIG. 4A.

FIGS. 5A to 5C are a series of schematic representations of a straightmicrofluidic channel in an isometric view (5A), a top view (5B), and aside view (5C) that show the formation of droplets containing, onaverage, a single particle from a fluid stream of ordered particleswithin the microfluidic channel.

FIG. 6 is a graph of data showing the fraction of droplets generatedwith an indicated number of particles per droplet.

FIGS. 7A and 7B are optical micrographs of random (FIG. 7A) and ordered(FIG. 7B) encapsulation of particles in a fluid. In FIGS. 7A-7B, thedroplets enclosed by a white circle have one particle per droplet; thedroplets enclosed by a square circle have two particles per droplet andthe droplets that are not surrounded by a white circle or square do notcontain a particle.

FIGS. 8A and 8B are optical micrographs showing encapsulated dropletformation using beads (FIG. 8A) or cells (FIG. 8B).

FIG. 9A shows the droplet formation process over time. FIG. 9B is aseries of three optical micrographs showing droplet formation usingbeads.

FIGS. 10A and 10B are graphs of data showing the fraction of dropletsthat contain a single particle and of droplets that contain more thanone particle as a function of the average number of particles perdroplet for (FIG. 10A) beads and (FIG. 10B) cells.

FIGS. 11A-11C are images of encapsulated cells were collected and flowedinto a wide microfluidic chamber in a largely uniform emulsion shown inbright field (FIG. 11A), green fluorescence (FIG. 11B), and redfluorescence (FIG. 11C).

DETAILED DESCRIPTION

Particles (e.g., cells or particles), in a fluid stream can beencapsulated in individual droplets by first forming an ordered streamof particles in the fluid stream within a microchannel and then formingthe fluid stream containing the ordered stream of particles intodroplets each containing, on average, a single particle. For example, afluid stream entering a droplet forming nozzle can contain twoevenly-spaced streams of particles (e.g., cells) whose longitudinalorder is shifted by half the particle-particle spacing. Ordering of theparticle within the fluid stream can occur when a high densitysuspension of particles (e.g., cells or particles) is forced to travelrapidly through a high aspect-ratio microchannel, where particlediameter is a large fraction (e.g., 10-40%) of the channel's narrowestcross-sectional dimension (i.e., a microfluidic channel having at leastone cross-sectional dimension that is about 2.5 to about 10 times thewidth of the largest dimension of the particles). This phenomenonprovides a method to controllably load single-cells into droplets,overcoming the intrinsic limitations set by Poisson statistics andensuring that a high percentage (e.g., 90% or more) of the dropletscontains exactly one cell.

Various microchannel configurations can be used to produce the orderedparticle fluid stream that can be used for droplet formation. Particlesorder laterally within the x-y plane (or cross-sectional plane) of thechannel and can also order longitudinally along the direction of flow.An additional dimension of rotational ordering can occur forasymmetrically shaped particles. The speed and number (or concentration)of the particles can be selected to provide a higher proportion ofdroplets containing a single particle than would be predicted by arandom statistical model absent the ordering of the particles within thefluid before droplet formation.

Unless otherwise indicated, as used herein, a “sample” refers to a fluid(e.g., gas or liquid) capable of flowing through a channel. Thus, asample can include a fluid suspension of biologically-derived particles(such as cells). The sample can comprise a material in the form of afluid suspension that can be driven through microfluidic channels can beused in the systems and methods described herein. For example, a samplecan be obtained from an animal, water source, food, soil, or air. If asolid sample is obtained, such as a tissue sample or soil sample, thesolid sample can be liquefied or solubilized prior to subsequentintroduction into the system. If a gas sample is obtained, it may beliquefied or solubilized as well. The sample may also include a liquidor gas as the particle. For example, the sample may comprise bubbles ofoil or other kinds of liquids or gases as the particles suspended in anaqueous solution. A sample can generally include suspensions, liquids,and/or fluids having at least one type of particle, cellular, droplet,or otherwise, disposed therein. Further, focusing can produce a flux ofparticles enriched in a first particle based on size. Exemplaryparticles can include, but are not limited to, cells, alive or fixed,such as adult red blood cells, fetal red blood cells, trophoblasts,fetal fibroblasts, white blood cells, epithelial cells, tumor cells,cancer cells, hematopoeitic stem cells, bacterial cells, mammaliancells, plant cells, neutrophils, T lymphocytes, B lymphocytes,monocytes, eosinophils, natural killer cells, basophils, dendriticcells, circulating endothelial cells, antigen specific T-cells, andfungal cells.

Samples can be diluted or concentrated to attain a predetermined ratiobefore and/or during introduction of the sample into the system. Ingeneral, the particle to volume ratio can be less than about 50%. Inother embodiments, particle to volume ratios can be less than about 40%,30%, 20%, 10%, 8%, or 6%. More particularly, in some embodiments,particle to volume ratios can be in a range of about 0.001% to about 5%,e.g., in a range of about 0.01% to about 4%. The ratio can also be inthe range of about 0.1% to about 3%, e.g., in the range of about 0.5% toabout 2%. In general, a maximum particle to volume ratio for a specifiedparticle size and channel geometry can be determined using the formula:

${{Max}\mspace{14mu}{Volume}\mspace{14mu}{Fraction}} = \frac{2N\;\pi\; a^{2}}{3{hw}}$

where N is the number of focusing positions in a channel, a is thefocused particle diameter, h is the channel height, and w is the channelwidth. The focusing position refers to a volume where the equilibriumpositions of flowing particles converge within a channel. A fluid samplecan be diluted or concentrated in batches before introduction into thechannel such that the sample ultimately introduced into the system hasthe required ratio before being introduced to the channel.

Particles suspended within a sample can have any size that allows themto be ordered and focused within the microfluidic channels describedherein. For example, particles can have a hydrodynamic size that is inthe range of about 40 microns to about 0.01 microns. For example,particles can have a hydrodynamic size that is in the range of about 20microns to about 0.1 microns; particles can also have a hydrodynamicsize that is in the range of about 10 microns to about 1 micron.

Various microfluidic systems and channel geometries can result inlongitudinally ordered particles in the direction of flow. Microchannelconfigurations for ordering a plurality of particles in a fluid streampassing through the microchannel can be designed based on certainparameters relating to the particle size and the microchanneldimensions, including the channel Reynolds number (Rc), the particleReynolds number (Rp), the Reynolds number based on mean channel velocity(Re), the particle hydraulic diameter (Dh) and the Dean Number (De).

The channel Reynolds number (Rc) describes the unperturbed channel flow:Rc=(UmDh)/v. The particle Reynolds number (Rp) includes parametersdescribing both the particle and the channel through which it istranslating: R_(p)=R_(c)(a²/D_(h) ²)=(U_(m)a²)/vD. Both dimensionlessgroups depend on the maximum channel velocity, U_(m), the kinematicviscosity of the fluid, and v=μ/ρ (μ and ρ being the dynamic viscosityand density of the fluid, respectively), and D_(h), the hydraulicdiameter, defined as 2wh/(w+h) (w and h being the width and height ofthe channel). The particle Reynolds number has an additional dependenceon the particle diameter, a.

The definition of Reynolds number based on the mean channel velocity canbe related to Rc by R_(e)=2/3R_(c). Channels with curvature createadditional drag forces on particles. When introducing curvature intorectangular channels, secondary flows develop perpendicular to thestreamwise direction due to the nonuniform inertia of the fluid. Twodimensionless numbers can be written to characterize this flow, the Deannumber (De) based on the maximum velocity in the channel, and thecurvature ratio (δ). The Dean number, De=Rc (D_(h)/2r)^(1/2) and thecurvature ratio, δ=D_(h)/2r, where r is the average radius of curvatureof the channel.

Using these parameters, microchannel dimensions and configurationsproviding focusing of particles within a fluid stream. Variouscombinations of these parameters will result in localization of a fluxof particles in a channel with a given channel geometry. Preferably,particles are passed through a microfluidic channel with a Rp of about 1or greater. In general, the Reynolds number of the flowing sample can beabout 1 to about 250, the Dean number of the flowing sample around acurved microchannel can be less than about 30, preferably about 20 orless, and/or the ratio of particle diameter to hydraulic diameter can beless than about 0.5. Channel cross-sections can include, but are notlimited to, square, rectangular, circular, triangular, diamond, andhemispherical. Particles of a predetermined size can be focused in eachof these exemplary cross-sections, and the equilibrium positions will bedependent on the geometry of the channel.

FIG. 1 is a graph of focusing results for particles as a function of twoparameters, hydraulic diameter (D_(h)) versus Dean number. The data inFIG. 1 was obtained by measuring the particle focusing of range ofparticle diameters (2-17 μm) and channel sizes (D_(h)=10-87 μm) over arange of Rc=0.075-225 for curving asymmetric channels. The focusingresults were plotted as a function of De and the ratio a/D_(h), as shownin FIG. 1. In FIG. 1, no particle focusing corresponds to filledsquares, focusing to two streams corresponds to open triangles, focusingto a single stream is represented by open circles, and more complexbehavior is shown as filled triangles. Data for this graph was collectedusing various size particles (2-17 μm) as well as four different channelgeometries, as described in co-pending patent application Ser. No.12/103,885 (filed Apr. 16, 2008), which is incorporated herein byreference in its entirety.

The results shown in FIG. 1 apply universally for any diameter ratio andDean number falling within a specific region independent of the specificgeometry. The data plotted in FIG. 1 appear similar to a phase diagramand are useful for determining the suitable microfluidic channels. Avertical movement on the diagram corresponds to changing particle sizeif channel geometries are held constant. To effect a focusing ofparticles in a fluid stream, one can choose a region in the phasediagram (i.e., a specific geometry) where a small change in particlesize leads to a change from a focused to an unfocused stream. Thus, oneparticle size is focused to a particular streamline and can be collectedas an enriched fraction, whereas the other, smaller, particles areunfocused. Numerous systems and methods for producing a focused streamof particles in a fluid stream are described in co-pending patentapplication Ser. No. 12/103,885 (filed Apr. 16, 2008), and areincorporated herein by reference in their entirety.

Particle size (at least over the range of cell diameters) had a minorrole in cross-stream ordering behavior. Inertial lift forces that leadto focusing of particles in the lateral dimension of the channel areknown to scale strongly with particle size, such that for the testedsystem cells and particles with diameters below ˜4 μm were seen to haveless robust ordering. For larger particles, the limitation is based onthe minimum channel dimension ˜27 μm. Generation of ordered streams forparticles above and below these limits (e.g. bacteria and plant cells)is expected to be possible by changing the channel dimensionsappropriately. Limitations could arise for scaling to smaller dimensionsas the pressure drop per unit channel length in the absence of particlesis approximately equal to

${32\mu\;{\overset{\_}{V}\left( \frac{w + h}{2{wh}} \right)}^{2}},$where μ is dynamic viscosity, V is the average fluid velocity and w andh are the channel width and height respectively.

Referring now to FIGS. 2A-2D, cell ordering in a rectangular geometrychannel for straight is described. The separation, ordering, andfocusing of particles can occur within these exemplary straightchannels. In general, at low flow rates, particles flowing within theseexemplary channels distribute uniformly along the length of a channelhaving (1) a cross-sectional aspect ratio (i.e., ratio of length towidth or vice versa) of about 1.5 to 8.0 (preferably about 1.5 to 4.0),and (2) a minimum cross-sectional dimension that is up to about 10 times(e.g., 2.5-10 times) the maximum cross-sectional dimension of a particlepassing through the channel in a fluid. In the illustrated embodiment,particles 9 μm in diameter suspended in water were observed in 50μm-wide square channels, providing a particle diameter to channeldiameter of 0.18. An inlet region is shown where the particles areinitially uniformly distributed within the fluid but start to focusshortly thereafter to the four channel faces. The degree of focusingincreases with Rp at a given distance along the channel and alsoincreases with the distance traveled along the channel. Preferably theparticle Reynolds number is about 1 or greater. For Rp=2.9 (Rc=90),complete focusing is observed after a distance of about 1 cm.

The cross-section of a straight channel 90 can be adjusted to producespecific and/or optimized focusing results. In particular, the aspectratio of the channel 90 cross-section can be changed from about 1 to 1to about 2 to 1 as shown in FIG. 2A. In addition, the particle diameterto channel diameter ratio is preferably greater than 0.3. When theaspect ratio and the particle diameter to channel diameter are adjustedin this way, less deviation in position is observed during particlefocusing in the channel 90. In addition, particles (e.g., cells) in thetwo ordering sites 94 a, 94 b are observed to interact and order acrossthe channel 90. Ordering can occur for low to high particleconcentrations, where only the particle-particle distance is affected byconcentration. Importantly, particles can become evenly spaced in thedirection of flow even to high particle concentration (e.g., 50×10⁶/Ml).

The ordering of particles, such as cells, in fluid passing through thechannel 90 provides a tighter distribution in particle lateral positionin the flow as well as improved particle-particle interactions leadingto long regular chains 92 of particles with uniform spacing in thedirection of flow, as shown in FIGS. 2B and 2C. Precision ordering ofcells and particles of 5-15 μm in size can be demonstrated for a varietyof particle/cell densities (<5%) at continuous flow, most clearlyillustrated in FIG. 2B. Further, particles ordered in positions acrossthe channel 90 also interact to create a uniform fluid buffer betweenthem.

In one exemplary system having a 2:1 rectangular cross-sectionalgeometry, particles all travel with a speed of 13.2-13.8 cm/s (meanfluid velocity being 11.9 cm/s) and exhibit a center-center spacing of42-45 μm between adjacent particles when they are focused to the sameside of the channel 90, but are separated by only 23-25 μm in thedirection of flow when the alternating pattern is present. These twopatterns can also be found in combination, the particular ratio of oneto the other depending most on the local concentration of particles; ifthe concentration is low, the particle-particle spacing present withinthe linear array is allowed, as shown in FIG. 2C. As the localconcentration increases, however, particles are found more frequently inthe interstitial sites on the other side of the channel 90, asillustrated in FIG. 2B. Equilibrium particle spacing at the end of a 6cm channel is generally linearly dependent on the particle diameter andchannel diameter.

In another example shown in FIG. 2D, the conditions described withrespect to FIGS. 2A-C are applied to particles of two differentpredetermined particle types. The particles 92 of a first type(illustrated as open circles) can be introduced into the channel througha first input branch (the lower branch), while a second particle type(illustrated as closed, shaded circles) can be introduced into thechannel through a second separate channel branch, the upper input branchas shown in FIG. 2D. As shown, the two types of particles move fromseparate input branches into a single channel and are ordered andfocused into two streams corresponding to two equilibrium positions onopposite sides of the channel. Where the first and second particles arediffering cell types, particles having differing chemistries, or somecombination thereof, having the particles focused and ordered such thatthe particles generally alternate between particles of the first typeand particles of the second type as they travel down the channel allowsfor greater opportunities to observe and manipulate interactions betweenparticles of the first and second types.

While the illustrated geometry for achieving the effects described withrespect to FIG. 2 has an aspect ratio of 1 to 1, similar fluid particleself-effects may be observed with other aspect ratios. In addition toratios of about 1 to 2, a reduction in symmetry can be observed inrectangular channels having dimensional ratios of approximately 15 to50, 3 to 5, and 4 to 5. Accordingly, the fluid particle self-orderingeffects can be seen for a dimensional aspect ratio of approximately 0.3(15/50) to a dimensional aspect ratio of approximately 0.8 (4/5), andthat the effects can be seen regardless of whether the longer dimensionis the width or the height.

Particles in a sample can also be ordered by passing the fluid samplethrough one or more symmetrically curved portions of a microfluidicchannel. In general, as Rc increases between 0.5 and 5, focusing intotwo streams of particles in the fluid can occur. As Rc increases, mixedstreams are again observed, in agreement with an increased contributionfrom Dean drag. FIG. 3 shows the separation, ordering, and focusing ofparticles in a fluid passing through a sserpentineal symmetricallycurved microfluidic channel. An aspect ratio of a serpentine channel canbe substantially 1 to 1 and/or can vary along a length thereof (e.g.,the aspect ratio of a sserpentineal channel can vary over the length ofthe channel between 1 to 1 and 2 to 1). Particles are randomlydistributed in the fluid at the inlet of the channel. As Rc increasesbetween 0.5 and 5, focusing into two streams of particles can occur. AsRc increases, mixed streams are again observed, in agreement with anincreased contribution from Dean drag.

The microchannel can also have one or more asymmetric curves, leading toa further reduction in the symmetry of particle focusing around theasymmetric curve region of the channel. In an asymmetrically shapedchannel, the net force generally acts in one direction, biasing a singlestable position of the initial distribution, and creating a singlefocused stream of particles. A time-averaged unidirectional centrifugaland/or drag force favors focusing down to a single stream betweenRe=1-15 Focusing becomes more complex as D e increases. Particles arefocused to one position of minimum potential with the addition ofcentrifugal forces or drag forces in the negative x-direction. Completefocusing can also occur for much smaller R p of about 0.15 and forshorter traveled distances (about 3 mm) than in the case of straightrectangular channels.

Various methods can be used for identifying ordered and focusedparticles within a channel. Labels or tags for identifying ormanipulating particles to be focused within the channels can beintroduced into the sample before, during, and/or after introduction ofthe sample into the system. Labeling or tagging of particles is wellknown in the art for use, for example, in fluorescence-activated cellsorting (FACS) and magnetic-activated cell sorting (MACS), and any ofthe various methods of labeling can be used. Exemplary labeling methodsand techniques are discussed in detail in U.S. Pat. No. 6,540,896entitled, “Microfabricated Cell Sorter for Chemical and BiologicalMaterials” filed May 21, 1999; U.S. Pat. No. 5,968,820 entitled, “Methodfor Magnetically Separating Cells into Fractionated Flow Streams” filedFeb. 26, 1997; and U.S. Pat. No. 6,767,706 entitled, “Integrated ActiveFlux Microfluidic Devices and Methods” filed Jun. 5, 2001; all of whichare incorporated by reference in their entireties.

Various techniques exist for moving the sample through a microfluidicchannel. For example, a microfluidic system can include a pumpingmechanism for introducing and moving the fluid sample into and throughone or more microfluidic channels. The pumping mechanism can alsoregulate and control a flow rate within the channels as needed. Aspecific pumping mechanism can be provided in a positive pumpingconfiguration, in a negative pumping configuration, or in somecombination of both. In one embodiment, a sample can be introduced intothe inlet and can be pulled into the system under negative pressure orvacuum using the negative pumping configuration. A negative pumpingconfiguration can allow for processing of a complete volume of sample,without leaving any sample within the channels. Exemplary negativepumping mechanisms can include, but are not limited to, syringe pumps,peristaltic pumps, aspirators, and/or vacuum pumps. In otherembodiments, a positive pumping configuration can also be employed. Asample can be introduced into the inlet and can be injected or pushedinto the system under positive pressure. Exemplary positive pumpingmechanisms can include, but are not limited to, syringe pumps,peristaltic pumps, pneumatic pumps, displacement pumps, and/or a columnof fluid. Oscillations caused by some pumping mechanisms, such as aperistaltic pump, can optionally be damped to allow for proper focusingwithin the channels.

Flow rates within the channels can be regulated and controlled. Forinstance, any number and variety of microfluidic valves can also beincluded in the system to block or unblock the pressurized flow ofparticles through the channels. The microvalve can include one or moremobile diaphragms or flexible membranes formed in a layer above achannel branch, inlet, or outlet such that upon actuation, the membraneis expanded up to decrease resistance within a channel branch, inlet, oroutlet, or expanded down into the channel to increase resistance withinthe same. Further details and discussion of such microfluidic diaphragmsare disclosed in PCT Publication No. PCT/US2006/039441 entitled,“Devices and Methods for Cell Manipulation” filed Oct. 5, 2007 andincorporated herein by reference in its entirety. Optionally, one ormore microfluidic, size-based separation modules or filters can beincluded to prevent clogging within the channels by preventing certainparticle sizes or particle types from entering the channels and/or tofacilitate collection of particles for downstream processing.

The fluid stream of ordered particles can pass through an outlet of amicrofluidic channel through a nozzle and into a medium suitable toinduce droplet formation from the fluid stream. Droplet formation of thefluid can be induced by injecting the fluid into a second immiscibleliquid, as described by Utada et al, Phys. Rev. Lett. 99, 094502 (2007),incorporated herein by reference in its entirety. The mechanism ofdroplet formation of the fluid is related to the presence of thesurrounding viscous liquid. A liquid forced through an orifice willultimately break into droplets at slow flows, whereas at faster flowsthe liquid forms a thin stream that breaks into droplets away from theorifice; these are the dripping and jetting regimes.

The transition between dripping and jetting in a two-phase coflowingstream. The behavior is characterized by a state diagram that depends onboth the capillary number of the outer fluid, C_(out), and the Webernumber of the inner fluid, W_(in); these parameters describe,respectively, the magnitude of the viscous shear forces from the outerliquid and the inertial forces from the inner liquid compared to surfacetension forces. A transition from the drop-dripping to jetting behavioris dependent on the capillary number of the outer pinching flow(C _(out)=η_(out) u _(out)/γ),and the Weber number for the inner flow,(W _(in)=ρ_(in) d _(tip) u _(in) ²/γ)where ρ is the density of the fluid, η is the viscosity, γ is thesurface tension between the two phases, d_(tip) is the diameter of theforming drop, and u is the fluid velocity. Both dimensionless numbersshould be below O(1) to be certain of stable dripping behavior. Usingthese parameters, the droplet diameter of the fluid can be calculatedbased on the composition of the fluid and the immiscible liquid intowhich the fluid introduced after passing through a nozzle. Dropformation is affected by parameters including the average velocities ofboth liquids, their viscosities and densities, surface tension, and thesurface chemistry and device geometry, as described by Utada et al,Phys. Rev. Lett. 99, 094502 (2007).

Within a straight microfluidic channel shown in FIG. 2A, hydrodynamicinteractions can cause particles to self-organize along one side of themicrochannel or into a diagonal/alternating pattern. The uniform spacingin the direction of flow (see side view) leads to the formation ofsingle particle droplets when the two lateral flows of oil pull dropletsfrom the aqueous stream (see isometric view) with the same (or higher)frequency that particles reach a microdrop generator (FIG. 5). As theresults for 0.89 beads per drop on average in FIG. 6 indicate, orderedencapsulation of beads (FIGS. 7B, 8B) generates more single-particledroplets (circles) and fewer empty (not marked) or multiple-particledroplets (boxes) than would have been possible from FIG. 7A stochastic(Poisson) loading. FIG. 7A shows stochastic encapsulation of beads froma disordered (random) stream of beads, in contrast to the results forordered encapsulation, FIG. 7B (46 out of 47 droplets contain a singlebead) resulting from an ordered stream of particles in the fluid priorto droplet formation. Scale bars correspond to 100 micrometers.

Favorable conditions generated two main classes of organized behavior inthe focusing channel preceding the drop generator (FIG. 5, top view):either (1) particles were focused into the same streamline along oneside of the channel, or (2) particles were arranged into an array thatalternated from one side of the channel to the other (FIG. 8A for beadsand FIG. 8B for cells).

FIG. 10A and FIG. 10B are graphs showing the fraction of droplets as afunction of particles (beads in FIG. 10A, cells in FIG. 10B). Each graphshows the resulting fractions of droplets that contain a single-particle(singles) and of droplets that contain multiple-particles (multiples)for concentrations between zero and one particles per drop. Nearlyperfect single-particle loading (maximum fraction of singles is λ) isachieved in all cases and the results are far superior to those expectedfrom Poisson statistics, especially for high concentrations. By fittingthe dependences of singles and multiples to linear functions of λ, theratio of singles to multiples can be calculated to be 30.9 for beads and56.5 for cells.

From high-speed video recording, it is evident that most multiplesresulted from particle aggregates that presumably formed when theinitial batches of suspension were concentrated. Therefore, the rate ofmultiples should be proportional to λ, consistent with FIG. 10A-10B,which are graphs of data showing the fraction of droplets that contain asingle-particle (singles) and of droplets that contain more than oneparticle (multiples) vs. average number of particles per drop (λ) for(FIG. 10A) beads and (FIG. 10B) cells. Data points (experiment) areplotted alongside curves expected for perfect (ordered) and random(Poisson) encapsulation. Fractions of singles fit a linear trend versusconcentration, where they occurred with a frequency of 0.937λ for beadsand 0.966λ for cells (1λ is ideal). Multiples should not occur forperfect ordering but resulted sporadically from pre-existing particleaggregates (0.0303λ and 0.0171λ for beads and cells, respectively). Eachpresented data point represents an analysis of 50-250 droplets in one ofmany wide-field frames of video, chosen at regular intervals fromhigh-speed videos up to 5½ min long, totaling 8.42×10³ beads in 18.9×10³droplets and 4.46×10³ cells in 21.6×10³ droplets. Moreover, perfectordering appears limited only by these preexisting particle aggregates.

The two patterns of self-organized behavior noted above shared threedistinctive characteristics: (1) each particle was separated from itsnearest neighbor by a uniform spacing in the direction of flow, (2)particles were always found near the side walls of the channel, and (3)particles moved only in the direction of flow as a group. To ensuresingle-particle droplets, the flow of oil can be adjusted to generatedroplets with a frequency not less than the frequency with which cellsin the more closely-spaced alternating pattern of order arrived at themicrodrop generator.

For example, to ensure that less than 2% of the occupied droplets aremultiples, so that any misleading cross-talk between cells in the samedrop is infrequent, Poisson statistics requires that cell suspension bediluted to λ=0.040, so that only 3.84% of all droplets contain asingle-cell. However, the methods presented herein can provide over 20times higher rates of single-cell droplets for the same ratio of singlesto multiples.

The methods and devices disclosed herein can be used to encapsulate avariety of biological materials, particularly cells, in fluid droplets.The ability to rapidly analyze and extract information from whole blood,for example, and its component cells is of great importance for medicaldiagnostics and applications in basic science. Blood cells themselvescontain an abundance of information relevant to disease, infection,malignancy, or allergy diagnosis. The methods and systems presentedherein relate to inertial microfluidic technology as a solution for highthroughput and precise microscale control of cell and particle motion.Methods and systems disclosed herein can be used for applications inblood cell subtype or rare cell enumeration, sorting, and analysis.Identification and analysis of rare cells, in particular, requires largesample sizes and high-throughput. The ability to sort, order, enumerate,and analyze particles continuously, differentially, and at high rates ina simple channel will be broadly applicable in a range of applicationsin continuous bio-particle separation, high-throughput cytometry, andlarge scale filtration systems.

The droplets can be formed using a nozzle in fluid communication withthe outlet of a microfluidic channel formed in a microfluidic chipdevice. A microfabricated chip can be provided and can have any numberand configurations of any of the channels described above formedtherein. A plurality of the channels formed in the microfabricated chipthat can be configured for receiving a sample introduced through theinlet and filter.

An analysis region can be provided in proximity to an output channels ofthe channels to monitor, sort, count, image, or otherwise analyze thefocused streams of particles (e.g., cells). The output channels can beprovided to receive and/or collect one or more focused streams ofparticles per channel after the streams travel through the analyticalregion of the chip. One or more output channels can also be provided forseparating particles of a predetermined type away from a main stream ofparticles via a microfluidic valve. A controller, which can include anynumber of hardware, software, and analytical elements can be included toassist in pre-sample processing, pumping, flow rate regulation, valveoperation, and any analysis to be performed on focused particles. Afterfocusing, particles can be collected from the output channels into areservoir or outlet for initial or additional analysis elsewhere, or fordisposal.

A variety of techniques can be employed to fabricate the chip havingchannels formed therein for the separation, ordering, and focusing ofparticles. In one particular embodiment, the chip can be formed of PMMA.The features, including channels, can be transferred onto anelectroformed mold using standard photolithography followed byelectroplating. The mold can be used to hot emboss the features into thePMMA at a temperature near its glass transition temperature (105° C.)under pressure (5 to 20 tons). The mold can then be cooled to enableremoval of the PMMA chip. A second piece used to seal the chip, composedof a similar or dissimilar material, can be bonded onto the first pieceusing vacuum-assisted thermal bonding. The vacuum prevents formation ofair gaps in the bonding regions. As will be appreciated by those skilledin the art, the chip can be formed of any material or combination ofmaterials as needed for specific pressure requirements within thechannels, as well as specific channel geometries and size requirements.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims. Methods ofmaking, analyzing, and characterizing some aspects of the polymerelectrolytes are described below.

Unless otherwise indicated, the materials described herein were used toperform the examples below. Fluorescent polystyrene microparticles(density ˜1.05 g/mL, 9.9 μm diameter, product #G1000) were purchasedfrom Duke Scientific (Fremont, Calif.). Particles were mixed to desiredweight fractions by dilution in Phosphate buffered saline (PBS) andstabilized by addition of 0.1% w/v Tween 20 (Sigma-Aldrich, St. Louis,Mo.). Cells (HL60 human promyelocytic leukemia cells, #CCL-240; ATCC,Manassas, Va.) were cultured in RPMI 1640 medium with 10% FBS andresuspended in PBS prior to use. A live-dead assay based on calcein AMand ethidium homodimer-2 (Invitrogen, Carlsbad, Calif.) was used todetermine cell viability/membrane integrity according to establishedprotocols.

For beads, FC-40 (3M, St. Paul, Minn.) were used with oil-phasesurfactant courtesy of RainDance Technologies (Lexington, Mass.), whoalso provided the fluorinated oil and PFPE-PEG block copolymersurfactant mixture (1.8% w/w in oil) used in the cell experiments. Aconcentration of 0.1% w/w Zonyl FSN-100 (DuPont, Wilmington, Del.) wasadded to the aqueous phase for cell experiments to reduce biologicalinteractions with the oil-water interface.

Microfluidic devices were fabricated using soft lithography techniques.SU-8 50 (MicroChem, Newton, Mass.) was spun at 2400 rpm for 30 secondsto create a 52 μm thick layer on a 10 cm silicon wafer. Thickness wasmeasured using a Dektak profilometer. The pattern wasphotolithographically defined in this layer using a mylar mask printedat 50,000 dpi. After development, PDMS (Sylgard 184; Dow Corning,Midland, Mich.) was poured onto the SU-8 master at a 10:1 ratio of baseto crosslinker, degassed in a vacuum chamber, and cured at 65° C.overnight. The devices were then cut from the mold; ports weresubsequently punched with a sharpened flat tip needle and devices werebonded to glass slides using oxygen plasma. After plasma treatment andplacement onto the glass substrate the devices were kept at 70° C. on ahotplate for 15 minutes to increase bonding. To ensure hydrophobicsurfaces throughout the microchannels, and thus allow the oil topreferentially wet the channel walls, the contents of a 1 mL syringe wasmanually forced, filled with air and a small amount of Aquapel (PPGIndustries, Pittsburgh, Pa.) inside the needle, through the channelnetwork until no residue was visible. Channel width was measuredoptically during operational conditions.

Example 1: Formation of an Ordered Stream of Cells

FIG. 4A illustrates focusing of blood cells in the same manner as rigidparticles. Five percent whole blood diluted in PBS is run throughrectangular channels of 50 μm width. At the outlet, 3 cm downstream,streak images of cells are observed in phase contrast. These appear asdark streams in the gray channel. The channel edges are also dark. As inthe case with rigid particles 3 streaks are observed which correspond tofour focus points on the rectangular channel faces.

FIGS. 4A and 4B illustrate focusing of cultured cell lines. As withparticles, deformable cells are focused to single streams. FIG. 4A showsstreak images of cells focusing for various R e numbers are shown. Theinlet of each focusing area is shown on the left. Focusing to a singlelane starts to occur for R e ^(˜)2 after 3 cm of travel. In FIG. 4B,intensity cross sections at various turns and at the outlet are shown.Note that at the outlet the width of the focused stream is comparable tothe diameter of a single cell (^(˜)15 μm).

Cell viability can be maintained during inertial focusing. Because cellstravel at high velocities (^(˜)0.5 m/s), it is important to evaluatecell viability and damage during this process. It should be noted thatcells traveling at steady state with the fluid experience only smallnormal and shear stresses over their surfaces, while significant forcesare briefly felt in the inlet and outlet regions where cells must beaccelerated by the fluid. In the systems described herein, the channelwidth at the inlet can optionally be gradually tapered to minimize thiseffect. High cell viability is found by vital stain after passingthrough an exemplary system. The scatter plot width and position forblood before processing appeared essentially unchanged after passingthrough the system. Cell debris and blebbing would produce a broaderdistribution of scatter.

No significant alterations in cell viability occur after they are passedthrough the inertial focusing systems described herein at high speeds.Even at average velocities of 0.5 m/s there was no discernable damage tocells (99.0% vs. 99.8% initial viability as measured by using afluorescent live/dead assay). High cell viability and throughput areimportant for applications such as flow cytometry.

Example 2: Encapsulation of Cells in Droplets

To demonstrate controlled single-cell microdrop generation, aflow-focusing geometry was used to emulsify concentrated suspensions ofHL60 cells or 9.9 lm-diameter polystyrene beads immediately after theyhad traversed a 27 lm-wide×52 lm-tall×6 cm-long rectangular microchannel(See, e.g., S. L. Anna, Applied Phys Lett, 82, 364-366 (2003),incorporated herein by reference in its entirety).

Bead or cell suspensions and oil were separately introduced into twosyringes and connected by either PEEK tubing (#1569; UpchurchScientific, Oak Harbor, Wash.) or Tygon tubing (TGY-010; Small Parts) tothe two inlets of the PDMS portion of the device. Outlets of PEEK tubingwere also connected to the outlet ports of the device and routed into awaste container or collection tube.

Flow was driven at constant volume rate by a syringe pump (PHD 2000;Harvard Apparatus, Holliston, Mass.). A glass syringe (1 mL; SGE,Austin, Tex.) with an inserted magnetic stir bar was utilized tomaintain well suspended solutions of particles prior to their injection,through a mechanism based on that reported recently where a steel ballbearing is moved magnetically within the syringe to induce mixing (R.Cooper and L. P. Lee, “Chips & Tips: Preventing suspension settlingduring injection,” Lab on a Chip, 21 Aug. 2007). A plastic syringe (1mL; BD, Franklin Lakes, N.J.) was used to drive oil with a flow rate of50-60 μL/min for bead experiments and 85 μL/min for cell experiments.The aqueous flow was set to 10 μL/min for beads and 13 μL/min for cells.If precautions were not taken, clogging can occur as the entiresuspension of particles funnels through one tiny orifice, the nozzle. Asimple solution can be used for the incorporation of a microfluidicfilter upstream of the focusing channel, at the site of the aqueousinlet. A series of narrow channels were included, slightly smaller inwidth than the narrowest point in the device, and they are arranged in aparallel fashion. Therefore, anything that enters the device that couldclog the narrow nozzle region will be caught in the filter thatimmediately precedes the long focusing channel, thus preventing thedevice from clogging catastrophically. A 0.2 μm PTFE syringe filter(09-720-7; Fisher Scientific, Pittsburgh, Pa.) was used for the oilinlet.

PDMS devices were mounted onto the stage of an inverted microscope(Nikon TE2000-U). High-speed camera imaging was conducted using whitelight in Köhler illumination with the focal plane. All neutral densityfilters were removed and the highest power on the lamp allowed imagingwith 2 μs exposures using a Phantom v4.2 camera (Vision Research, Wayne,N.J.). Frame intervals from 62.5 μs-100 ms were used.

Random arrangements of particles enter the inlet of the system and aftertraversing the ordering channel arrive at the droplet generator portionof the microfluidic system well ordered. The oil flow rate was tuned tomatch the frequency of drop generation with that of the ordering. Thechannel is 27 μm wide and particles are 10 μm in diameter.

For the experimental conditions employed, the center-to-center spacingbetween adjacent particles focused on the same side of the channel was48.2±4.0 micrometers for beads and 33.5±3.7 micrometers for cells,corresponding to velocities of 13.7±0.1 cm/s and 20.6±0.7 cm/s,respectively. In comparison, the longitudinal spacing between particlesthat self-organized into the alternating pattern was reduced to 24.4±1.0micrometers for beads and 19.0±2.0 micrometers for cells, correspondingto velocities of 13.70±0.04 cm/s and 21.1±0.3 cm/s, respectively.

The current system was operated at some of the fastest possible dropgeneration rates (˜15 kHz). The system operated at this rate because theordering phenomenon functions more robustly at higher channelvelocities. The system is limited from going above this rate muchfurther without changes in channel geometry due to a transition from thedrop-dripping to jetting behavior. In the current system, furtherincreases in drop generation rates could be achieved by tuning theseparameters (e.g. by reducing surfactant concentrations to increasesurface tension). Decreasing the viscosity of the outer flow could alsoyield dripping behavior at drop generation rates exceeding 15 kHz.

To ensure single-particle droplets, the flow of oil was adjusted togenerate droplets with a frequency not less than the frequency withwhich cells in the more closely-spaced alternating pattern of orderarrived at the microdrop generator. More precisely, the flow of oil wasset to 50-60 microliters/min during bead experiments and 85microliters/min during cell experiments, causing 21.7 pL droplets toform at 7.7 kHz for beads (see FIG. 9B) and 14.6 pL droplets to form at14.9 kHz for cells (see FIG. 9A).

To confirm that the high aqueous flow rate required to induceself-ordering (aqueous flow rate was 10 microliter/min for beads and 13microliter/min for cells) did not adversely affect the cells, theirsurvival rates were tested after encapsulation and found that 92.9% ofcells retained membrane integrity, as compared with 96.2% for controls(see FIG. 11A-C). Encapsulated cells were collected and flowed into awide microfluidic chamber. Images of cells in the largely uniformemulsion are shown in bright field (a), green fluorescence (b), and redfluorescence (c). Exposure time was 500 ms for green fluorescence and 10sec for red fluorescence. Cells were stained with a live/dead stainprior to encapsulation in the system. Cells that lost membrane integrityduring the cell preparation process prior to entering the chip appearbright red (c), while cells with membranes disrupted during thesingle-cell encapsulation process leak green viability dye throughoutthe drop in which they were encapsulated (b). Live cells have higherintensity green signals (b) that did not spread throughout the drop inquestion. Scale bars: 100 μm.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

The invention claimed is:
 1. A method of ordering, identifying, andencapsulating particles in droplets, the method comprising flowing aplurality of particles in a fluid sample through a channel having aminimum cross-sectional dimension D, wherein a largest particle in theplurality has a maximum cross-sectional dimension that is at least about0.1 D, wherein certain of the plurality of particles may be labeled witha tag; controlling a flow rate of the fluid sample within the channel tohave a channel Reynolds number (Re) of about 1 to about 250 to form asubstantially evenly spaced, longitudinally ordered stream of particlesin the fluid sample within the channel; identifying particles labeledwith a tag in the stream of particles; manipulating identified particleslabeled with a tag to exit the stream of particles and flow in a portionof the fluid sample one at a time through a nozzle; and contacting thefluid sample containing identified particles labeled with a tag exitingthe nozzle with a fluid medium that is immiscible with the fluid sampleat a velocity effective to generate individual liquid droplets of fluidsample dispersed within the fluid medium, wherein the liquid dropletsexit the nozzle and wherein the flow rates of the fluid sample and fluidmedium are controlled to produce a set of droplets in which a fractionof single-particle droplets is higher than a corresponding fraction ofsingle-particle droplets predicted by Poisson statistics.
 2. The methodof claim 1, further comprising labeling certain of the plurality ofparticles with a tag.
 3. The method of claim 1, wherein the tag is afluorescent tag.
 4. The method of claim 3, wherein identifying andmanipulating the particles labeled with a tag comprisesfluorescence-activated cell sorting.
 5. The method of claim 1, whereinthe tag is a magnetic tag.
 6. The method of claim 5, wherein identifyingand manipulating the particles labeled with a tag comprisesmagnetic-activated cell sorting.
 7. The method of claim 1, wherein thefluid medium flows to the nozzle through two or more lateral channelsconnected to the nozzle.
 8. The method of claim 1, wherein the particleReynolds number is at least about
 1. 9. The method of claim 1, whereinthe particles are of substantially the same size and the largestparticles in the plurality have a maximum cross-sectional dimension thatis about 0.1 D to 0.4 D.
 10. The method of claim 1, wherein theparticles are cells.
 11. The method of claim 1, wherein the particleslabeled with a tag are cells.
 12. The method of claim 11, wherein thecells are one or more of adult red blood cells, fetal red blood cells,trophoblasts, fetal fibroblasts, white blood cells, epithelial cells,tumor cells, cancer cells, hematopoietic stem cells, bacterial cells,mammalian cells, plant cells, neutrophils, T lymphocytes, B lymphocytes,monocytes, eosinophils, natural killer cells, basophils, dendriticcells, circulating endothelial cells, antigen specific T-cells, andfungal cells.
 13. The method of claim 1, wherein the liquid sample is anaqueous medium and the liquid medium is an oil.
 14. The method of claim1, wherein the fluid sample is passed through the channel at a rate ofat least about 10 microliters/minute.
 15. The method of claim 1, whereinthe fluid passes through a curved channel portion having a Dean numberof up to about
 30. 16. The method of claim 1, wherein the rate ofdroplet formation has a frequency that is not less than the frequencywith which the particles in the fluid sample leave the channel.
 17. Themethod of claim 1, wherein the particle Reynolds number is at leastabout
 1. 18. The method of claim 1, wherein the average volume of one ofthe plurality of droplets is about 1-100 pL.
 19. The method of claim 1,wherein the droplets are formed at a rate of about 1-20 kHz.
 20. Themethod of claim 1, wherein the flow rate of the fluid sample and theflow rate of the fluid medium are controlled such that at least 80% ofthe set of droplets contain only one particle and fewer than 20% of theset of droplets contain zero particles.